Field of the invention

The present invention relates to the field of chemical mutagenesis of cells. In particular, the present invention is in the field of chemical mutagenesis of M. esculenta cells, preferably friable embryogenic callus cells (FECs).

Background

Cassava ( Manihot esculenta) is a crop commonly grown in South America, Sub-Saharan Africa and Asia. It is grown primarily for its starchy storage roots which are used as a food source or as a source of starch for a multitude of industrial purposes. Cassava breeding is known to be a difficult and time consuming process (e.g. due to low flowering rates, low seed yields, inbreeding depression, limited knowledge of trait inheritance, few molecular markers, etc.). Therefore, the ability to introduce new and useful traits into cassava is limited. New traits of interest to agriculture and industry include (but are not limited to) herbicide resistance, increased storage, root yield, disease resistance, early flowering and modified starch properties.

Physical and chemical mutagenesis are well-known methods for introducing genetic variability into an organism, resulting in new traits in the mutant offspring. Mutant plants derived from such mutagenesis are not considered genetically modified organisms, thus new varieties developed using this method are not subject to GMO regulations and expensive, time-consuming consultation processes before approval for field release and commercial production.

Jain SM (Gene Conserve (2007), 6 (23): 329-343) teaches that both chemical and physical mutagens can be used to induce mutations. Among them, gamma rays and ethyl-methane sulfonate (EMS) are widely used for mutation induction. Physical and chemical mutagenesis has been suggested previously for cassava (e.g. Mba C et al (2010). Plant Cell Culture: Essential Methods. Ch.7: 1 1 1-130; Ahiabu RKA et al (1997), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Kenya, XA9745173; Tadele et al. Molecular Techniques in Crop Improvement: 2nd Edition (2009). Ch. 13: 307-332).

In addition, the art teaches the mutation of cassava using gamma radiation of fine embryogenic cell suspension cultures (Jain SM (supra) ), young leaf lobes, somatic embryos or cotyledonary segments from somatic embryos (Joseph R et al (2004) Plant Cell Rep (2004) 23:91- 98). However compared to chemical mutagenesis, the use of physical mutagens result in less point mutations and in more chromosomal damage. In addition physical mutagenesis, such as radiation, requires the use of specialized facilities and in practice radiation conditions are often hard to control. These drawbacks thus limit the use of physical mutagens in cassava.

In addition, physical and chemical mutagenesis has been described in cassava using shoot meristems (e.g. Ahiabu RK et al (1997), Mutagenesis for ACMV resistance in a Ghanian cassava cultivar 'Bosom Nsia', XA9744546) which are complex multi-cellular structures containing both differentiated and undifferentiated meristematic tissues. Mutagenesis of shoot meristems is likely to produce high numbers of chimeric mutant offspring where adult offspring contain a random mixture of mutant and non-mutant cells. Thus, the expression and penetration of the new trait is unpredictable and unstable.

Hence, there is still a strong need in the art for an effective method to introduce random point mutations in cassava. In addition, there is a need for the effective method to introduce such point mutations in cassava without the generation of chimeric plants.

Summary of the invention

In a first aspect, the invention pertains to a method for the production of a Manihot esculenta cell having a mutagenized DNA, wherein the method comprises the step of contacting the cell with the mutagenic chemical N-ethyl-N-nitrosourea (ENU), and wherein preferably the DNA is genomic DNA.

Preferably, the mutagenic chemical introduces a SNP into the DNA of the cell, wherein preferably the number of introduced SNPs is at least 0.004 SNPs per kb.

Preferably, the number of introduced SNPs is between about 0.001 - 0.01 SNPs per kb, wherein more preferably the number of introduced SNPs is between about 0.004 - 0.006 SNPs per kb.

In a preferred embodiment, the Manihot esculenta cell is a callus cell, wherein preferably the callus cell is an embryogenic callus cell and wherein more preferably the Manihot esculenta cell is a friable embryogenic callus (FEC) Manihot esculenta cell.

In a preferred method of the invention, the cell is incubated in a mutagenic solution comprising the mutagenic chemical and wherein the concentration of the mutagenic chemical in the solution is between about 0.01 % - 10% (w/v), preferably between about 0.1 % - 2% (w/v).

Preferably, the mutagenic solution comprises at least 70% liquid GDS medium, preferably at least 90% liquid GDS medium, and the mutagenic chemical.

In a further preferred embodiment, the cell is contacted with the mutagenic chemical for a period of about 10 - 200 minutes, preferably for a period of about 100 - 150 minutes, and wherein preferably the period is a contiguous period.

Preferably, the cell is contacted with the mutagenic chemical at a temperature of about 5 - 50 °C, preferably 15 - 25 °C. Preferably, the cell is contacted with the mutagenic chemical at a temperature that is maintained at about 5 - 50 °C, preferably 15 - 25 °C, during the period that the cell is contacted with the chemical. In a preferred embodiment, the method further comprises a step of generating a cotyledonary embryo from the mutagenized cell.

Preferably, the method comprises a further step of generating a plantlet and/or a plant.

In a preferred embodiment, the method further comprises detecting at least one mutation in the DNA and optionally assigning a phenotype to the detected mutation.

In a second aspect, the invention relates to a method for chemical mutagenesis of DNA in a Manihot esculenta cell, wherein the method comprises a step of contacting the cell with the mutagenic chemical 1 -Ethyl-1 -nitrosourea (ENU), and wherein preferably the DNA is genomic DNA.

In a third aspect, the invention concerns a mutagenized Manihot esculenta cell, cotyledonary embryo, plantlet and/or plant that is obtainable by the method as defined herein.

In a fourth aspect, the invention pertains to a mutagenic solution comprising i) the mutagenic chemical 1 -Ethyl-1 -nitrosourea ii) a Manihot esculenta cell.

In a fifth aspect, the invention concerns the use of the mutagenic chemical 1 -Ethyl-1 -nitrosourea for the chemical mutagenesis of the DNA in a Manihot esculenta cell.

Definitions

Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al.. Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al.. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

The singular terms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a combination of two or more cells, and the like. The indefinite article "a" or "an" thus usually means "at least one". The term“and/or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

As used herein, the term“about” is used to describe and account for small variations. For example, the term can refer to less than or equal to ± (+ or -) 10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1 %, less than or equal to ±0.5%, less than or equal to ±0.1 %, or less than or equal to ±0.05%. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

The term“comprising” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

"Plant" refers to either the whole plant or to parts of a plant, such as cells, tissue or organs (e.g. pollen, seeds, gametes, roots, leaves, flowers, flower buds, anthers, fruit, etc.) obtainable from the plant, as well as derivatives of any of these and progeny derived from such a plant by selfing or crossing.

"Plant cell(s)" include protoplasts, gametes, suspension cultures, microspores, pollen grains, etc., either in isolation or within a tissue, organ or organism.

An "explant" is a piece of tissue taken from a donor plant for culturing.

A "meristem" or "meristematic center" is a group of tissue forming cells capable of further development into plant organs; e.g., shoots and roots.

"Somatic embryogenesis" is the process, preferably using tissue culture techniques, for generating multiple embryos from an explant. The embryos from a given tissue source are preferably genetically identical.

Detailed description of the invention

The current invention concerns a method for chemical mutagenesis of cassava utilising friable embryogenic callus (FECs), which are undifferentiated cells derived from adult cells that are capable of undergoing embryogenesis and formation of an entirely new plant. The results from transformed plants regenerated from FECs demonstrate that formation of chimeric plants is rare. The invention therefore represents an important advance for large-scale cassava mutagenesis and trait development.

The inventors further discovered that chemical mutagenesis of cassava ( Manihot esculenta) is hampered by toxicity. In particular, many chemical mutagens are highly toxic and lead to cell death of FECs at low concentrations. Surprisingly, the mutagen N-ethyl-N-nitrosourea (ENU) did not result in severe toxicity, i.e. resulted in the least toxicity of the mutagens tested under the conditions used. In addition, exposure to ENU caused a significantly increased mutation frequency and resulted in a broad mutation spectrum in comparison to e.g. the well-known mutagen EMS.

Hence in a first aspect, the invention concerns a method for the production of a plant cell having a mutagenized DNA, wherein the method comprises the step of contacting the cell with a mutagenic chemical. Preferably, the plant cell is a Manihot esculenta cell.

Preferably, the plant cell remains viable before, during and after the mutagenesis step. Preferably, the cell can be propagated after the mutagenesis step.

The plant cell contacted with the mutagenic chemical can refer to a plant cell in isolation or in tissue culture, or to a plant cell contained in a plant or in a differentiated organ or tissue. Hence, a reference to a plant cell in the description or claims is not meant to refer only to isolated cells or protoplasts in culture, but refers to any plant cell, independent of its location or cell type.

The plant cell that is contacted with the mutagenic chemical ENU is preferably a wild-type Manihot esculenta cell. It is understood herein that a wild-type cell may be a plant cell from a plant occurring in nature as well as a plant cell from a cultivar. A wild-type cell within this disclosure is to be understood the cell prior to the application of the method of the invention. Preferably, the wild- type plant cell is not a transgenic plant cell. The M. esculenta cell can be obtainable or obtained from a cassava variety selected from the group consisting of TMS60444, TME7, TME3, TME14, TME204, T200, Ebwanatereka, Kibandameno and Serere, preferably obtained from The International Institute of Tropical Agriculture (IITA, IITA Headquarters, PMB 5320, Oyo Road, Ibadan 200001 , Oyo State, Nigeria). Preferred cassava varieties are TMS60444 or TME7.

The M. esculenta cell can also be a genetically modified M. esculenta cell. The genetically modified cell refers herein to a plant cell having one or more genetic modifications, e.g. alterations, insertions and/or deletions. A genetically modified M. esculenta cell can comprise an exogenous gene or additional copy or copies of an endogenous gene, said exogenous gene or additional endogenous gene can be integrated into the genome. The genetically modified M. esculenta cell can be obtained by introgression of a certain gene or can be obtained by transformation with an (isolated) polynucleotide sequence. Methods for obtaining introgressed / transgenic plant cells and plants are well known in the art.

Methods for transformation of a plants cell include but are not limited to Agrobacterium- mediated transformation of plant cells, particle bombardment of plant cells, transformation of plant cells using whiskers technology, transformation using viral vectors, electroporation of plant protoplasts, direct uptake of DNA by protoplasts using polyethylene glycol, microinjection of plant explants and/or protoplasts. Agrobacterium-mediated transformation is a preferred method to introduce the nucleic acid molecule of the invention into plant cell.

The mutagenized DNA is mutagenized in comparison to the same DNA before exposure to the mutagenic chemical ENU. As a non-limiting example, a plant cell culture can be obtained and DNA of part of the cells can be analysed prior to mutagenesis, while part of the cells are mutagenized and the DNA is analysed after the mutagenesis. The DNA from before and after the mutagenesis step can be compared. Alternatively or in addition, the mutagenized DNA can be compared to a reference genome and the reference genome can be a reference Manihot esculenta genome.

The DNA that is mutagenized is at least one of genomic DNA, chloroplast DNA and mitochondrial DNA. In a preferred embodiment, the mutagenized DNA is at least genomic DNA.

The mutagenic chemical is preferably an alkylating agent. Preferably, the mutagenic chemical is an alkylating agent that acts by transferring an ethyl group to nucleobases in nucleic acids. Preferably the mutagenic chemical is selected from the group consisting of N-ethyl-N- nitrosourea (ENU), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), sodium azide (NaN3), azacytidine (AzaC) and 4-nitroquinoline 1-oxide (NQO). In a preferred embodiment, the mutagenic chemical is N-ethyl-N-nitrosourea (ENU).

Mutagenesis is herein understood as the introduction of point mutations in the DNA. Preferably, these point mutations are introduced at random locations in the DNA. The mutagenic chemical may introduce all mutation types. The introduced point mutations are at least one of A to C, A to G, A to T, C to A, C to G, C to T, G to A, G to C, G to T, T to A, T to C and T to G. Preferably, the point mutations introduced by the mutagenic chemical are at least one of A to C, A to G, A to T, C to A, C to T, G to A, G to T, T to A, T to C and T to G. The point mutations introduced by the mutagenic chemical may or may not cause C to G and/or G to C mutations. Preferably, the mutagenic chemical introduces all mutation types.

The introduced point mutations are preferably single-nucleotide polymorphisms (SNPs). In a preferred embodiment, the method of the invention therefore comprises a step of contacting an M. esculenta cell with a mutagenic chemical, wherein the mutagenic chemical introduces a SNP into the DNA of the cell. In a preferred embodiment, the number of SNPs is between about 0.0005

- 0.1 SNPs per kb, or about 0.001 - 0.09, about 0.001 - 0.08, about 0.001 - 0.07, about 0.001 - 0.06, about 0.001 - 0.05, about 0.001 - 0.04, about 0.001 - 0.03, about 0.001 - 0.02 or about 0.001

- 0.01 SNPs per kb. Preferably, the number of introduced SNPs is between about 0.001 - 0.01 SNPs per kb. Preferably, the number of introduced SNPs is between about 0.002 - 0.01 , about 0.003 - 0.009, about 0.004 - 0.009, about 0.004 - 0.008, about 0.004 - 0.007 or about 0.004 - 0.006 SNPs per kb. Preferably, the number of introduced SNPs is between about 0.004 - 0.008, preferably 0.004 - 0.006 SNPs per kb.

In a further embodiment, the number of introduced SNPs is at least 0.001 SNPs per kb. The number of introduced SNPs can be at least 0.001 , 0.002, 0.003 or 0.004 SNPs per kb. Preferably, at least 0.004 SNPs per kb.

The number of SNPs are determined per kb, i.e. per kilobase of genomic sequence, i.e. the mutation frequency. For example, 2 SNPs per kb is herein understood as 2 SNPs per 1000 consecutive bases of the genomic sequence.

In another embodiment, the Manihot esculenta cell is a Manihot esculenta callus cell. A plant callus is a growing mass of unorganized plant parenchyma cells. Hence, a callus cell is preferably an unorganized plant parenchyma cell, which is or can be part of a callus. Callus can be produced from a single differentiated cell, and callus cells can be totipotent, being able to regenerate the whole plant body. The plant callus is preferably derived from a somatic tissue or tissues, preferably a tissue that is available for explant culture. The cells that give rise to callus and somatic embryos preferably undergo rapid division and/or are partially undifferentiated such as meristematic tissue.

Callus cultures are often broadly classified as being either compact or friable. Friable calluses preferably fall apart easily, and can e.g. be used to generate cell suspension cultures. A friable callus can be generated according to any method known in the art, for example as described in Danso et al (Optimisation of somatic embryogenesis in Cassava; International Atomic Energy Agency (2017), J. Jankowicz-Cieslak et al (eds.), Biotechnologies for Plant Mutation Breeding. Chapter 5:p73-89).

Callus may directly undergo direct organogenesis and/or embryogenesis where the cells will preferably form an entirely new plant. The callus cell used in the method of the invention is preferably friable or compact, more preferably friable. In addition or alternatively the callus cell is rooty, shooty, or embryogenic callus (Ikeuchi M, Plant Cell. 2013 Sep; 25(9): 3159-3173).

In one embodiment, the cell is an embryogenic callus cell, e.g. an M. esculenta embryogenic callus cell. An embryogenic callus cell is preferably characterized in that the callus cell is capable of forming at least a cotyledonary embryo. Preferably, the embryogenic callus cell can generate a new plant through the process of somatic embryogenesis (Zimmerman JL, The Plant Cell, Vol. 5, 141 1-1423). In a further embodiment, the embryogenic callus cell is a compact embryogenic callus cell or afriable embryogenic callus cell. Preferably, the cell for use in the method of the invention is a friable embryogenic callus (FEC) cell, preferably a Manihot esculenta friable embryogenic callus cell.

The callus cell for use in the method of the invention can be broken apart from a cluster to generate smaller parts or small FEC cell clumps prior to the mutagenesis step. In a preferred embodiment, the plant callus cell remains in contact to, e.g. adheres to, other callus cells during the mutagenesis step. Hence, the FEC cell can remain part of a small clump of cells during the mutagenesis step.

The FECs for use according to the invention can be generated and multiplied according to any method known in the art, for example as described in e.g. Bull SE et al (Nature Protocols (2009), 4(12): 1845-54) or Chauhan RJ et al (Plant Cell, Tissue and Organ Culture (2015), 121 (3): 591— 603).

In an embodiment of the invention, the cell as defined herein is incubated in a mutagenic solution comprising the mutagenic chemical. The mutagenic chemical is preferably any mutagenic chemical as defined herein. The mutagenic solution can comprise more than one type of mutagenic chemical, e.g. at least two, three or four mutagenic chemicals. Preferably the mutagenic solution comprises at least one mutagenic chemical and wherein preferably, the mutagenic chemical is ENU. Alternatively, the mutagenic solution can comprise only one mutagenic chemical, wherein the only one mutagenic chemical is ENU.

The concentration of the mutagenic chemical in the mutagenic solution is preferably sufficient to introduce a SNP in the DNA of the plant cell, when the cell is incubated in the mutagenic solution under the conditions as defined herein below. Preferably, the concentration of the mutagenic solution is sufficient to introduce SNPs in the DNA at a frequency as defined herein above. In addition or alternatively, the concentration of the mutagenic chemical preferably does not result in cell death of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or about 100% of the plant cells, when these cells are incubated with the mutagenic solution under the conditions as defined herein below.

In an embodiment, the final concentration of the mutagenic chemical in the mutagenic solution is at least about 0.001 %, 0.005%, 0.01 %, 0.03%, 0.05%, 0.08%, 0.1 %, 0.3%, 0.5%, 0.6%, 0.8%, 1.0%, 1.3%, 1.5%, 1.8%, 2.0%, 2.5%, 3.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25% or 30% (w/v) The concentration of the mutagenic chemical in the mutagenic solution is preferably less than 30%, 25%, 20%, 15%, 10%, 7.0%, 5.0%, 4.0%, 3.0%, 2.5% or 2.0% (w/v). In an embodiment, the concentration of the mutagenic chemical in the mutagenic solution is preferably about 0.001 %, 0.005%, 0.01 %, 0.03%, 0.05%, 0.08%, 0.1 %, 0.3%, 0.5%, 0.6%, 0.8%, 1.0%, 1.3%, 1.5%, 1.8%, 2.0%, 2.5%, 3.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25% or 30% (w/v). The term w/v is herein understood as gram of the mutagenic chemical per ml_ of the mutagenic solution ( i.e . g/ml * 100%).

In an embodiment, the concentration of the mutagenic chemical in the mutagenic solution is between about 0.001 % - 30% (w/v), 0.005 - 25%, 0.01 - 10%, 0.03 - 7.0%, 0.05 - 5.0%, 0.08 - 4.0%, 0.1 - 3.0%, 0.1 - 2.0%, 0.3 - 2.0%, 0.5 - 2.0%, 0.6 - 2.0 %, 0.8 - 2.0 %, 1 .0 - 2.0%, 1.3 - 2.0%, 1.5 - 2.5%, 1.8 - 3.0%, 2.0 - 3.0% or 2.5 - 3.0% (w/v), preferably between about 0.01 % - 10% (w/v), preferably between about 0.1 % - 2% (w/v), and preferably between about 0.08% - 1.0% (w/v)

The mutagenic solution for use according to the invention as defined herein can be any solution comprising the mutagenic chemical. Preferably, the mutagenic solution is a freshly prepared mutagenic solution.

The mutagenic solution can comprise any suitable solution known in the art, such as, but not limited to, an isotonic and/or a buffered solution or a cell culture medium. Preferably, the mutagenic solution does not contain a component that can partly of fully inactivate and/or react with the mutagenic chemical.

A preferred buffer solution is e.g. PBS (phosphate-buffered saline). A preferred medium is liquid GDS medium. Hence in an embodiment, the mutagenic solution comprises liquid GDS medium in combination with the mutagenic chemical. Liquid GDS medium is known to the skilled person and described e.g. in Bull SE et al (supra). Liquid GDS medium can contain the following components: about 2.7 g/L Gresshof and Doy salts, about 20 g/L sucrose, about 12 mg/L picloram and a pH of about 5.8.

In an embodiment of the invention, the concentration of buffer solution, such as PBS, in the mutagenic solution is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% (v/v).

In an embodiment of the invention, the concentration of medium, such as liquid GDS medium, in the mutagenic solution is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% (v/v). In an embodiment, the mutagenic solution can consist of liquid GDS medium, and a mutagenic chemical. Hence, in this embodiment the mutagenic chemical is dissolved in liquid GDS medium. M. esculenta plant cells as defined herein can be subsequently be added to the mutagenic solution as defined herein.

In a further embodiment, the mutagenic solution comprises at least 70% liquid GDS medium, preferably at least 90% liquid GDS medium, and the mutagenic chemical. In another embodiment, at least one, two, three or all media types used in the method of the invention are described in Bull SE et al (supra) or Chauhan RJ et al (supra)

In the method of the invention, the M. esculenta plant cell is contacted with the mutagenic chemical under conditions that allow for mutagenesis of the DNA. Preferably the plant cell is contacted with the mutagenic chemical under conditions that result in a DNA mutation frequency as detailed above. The mutation frequency can be dependent on the concentration of the mutagenic chemical as detailed above. Further conditions that can influence the DNA mutation frequency and/or cell toxicity are the exposure time and the temperature.

Hence in an embodiment of the invention, the plant cell is contacted with the mutagenic chemical for a period of time that is sufficiently long to introduce SNPs in the DNA at a mutation frequency as defined herein above. The period of time that the plant cell is contacted with the mutagenic chemical preferably does not result in cell death of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or about 100% of the plant cells.

It is contemplated within the invention that the plant cell is contacted at least 1 , 2, 3, 4 or 5 times with the mutagenic chemical, e.g. to reduce the build-up of toxic breakdown products. The total time period the plant is contacted with the mutagenic chemical is a period of at least about 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 200, 220, 240, 260, 280 or 300 minutes. In addition or alternatively, the cell is contacted with the mutagenic chemical for a total period that is less than about 500, 450, 400, 350, 300, 280, 260, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 1 10, 100, 90, 80, 70, 60, 50, 40 or 30 minutes. In a further embodiment, the total time period the plant is contacted with the mutagenic chemical is about 1 , 2, 3,4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 200, 220, 240, 260, 280 or 300 minutes.

The plant cell can be contacted several times with the mutagenic chemical. Preferably the plant cell is contacted only once with the mutagenic chemical and hence the total period is a single, contiguous period.

In a further embodiment, the plant cell is contacted with the mutagenic chemical for a total time period of about 1 - 500, about 5 - 450, about 10 - 400, about 20 - 350, about 30 - 300, about 40 - 250, about 50 - 200, about 40 - 200, about 30 - 200, about 20 - 200 or about 10 - 200 minutes. Preferably for a total period of about 80 - 170, about 90 - 160 or about 100 - 150 minutes. Preferably, about 15 - 150 minutes or about 30 - 120 minutes.

Preferably, the plant cell is contacted with the mutagenic chemical for a total time period of about 5 - 80, about 10 - 60 or about 20 - 40 minutes. Preferably, the plant cell is contacted with the mutagenic chemical, wherein the concentration of the mutagenic chemical in the mutagenic solution is between about 0.05% - 2% (w/v) for a total time period of about 15 - 150 minutes.

Preferably, the plant cell is contacted with the mutagenic chemical, wherein the concentration of the mutagenic chemical in the mutagenic solution is between about 0.08% - 1.0% (w/v) for a total time period of about 15 - 150 minutes.

Preferably, the plant cell is contacted with the mutagenic chemical, wherein the concentration of the mutagenic chemical in the mutagenic solution is between about 0.5% - 0.7% (w/v) for a total time period of about 15 - 45 minutes.

Preferably, the plant cell is contacted with the mutagenic chemical, wherein the concentration of the mutagenic chemical in the mutagenic solution is about 0.6% (w/v) for a total time period of about 30 minutes.

Preferably, the plant cell is contacted with the mutagenic chemical, wherein the concentration of the mutagenic chemical in the mutagenic solution is between about 0.08% - 0.5% (w/v) for a total time period of about 100 - 150 minutes.

Preferably, the plant cell is contacted with the mutagenic chemical, wherein the concentration of the mutagenic chemical in the mutagenic solution is about 0.1 % (w/v) for a total time period of about 120 minutes.

In a further embodiment of the invention, the plant cell is contacted with the mutagenic chemical at a temperature that is suitable for introducing SNPs in the DNA at a mutation frequency as defined herein above. The temperature at which the plant cell is contacted with the mutagenic chemical preferably does not result in cell death of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or about 100% of the plant cells.

A suitable temperature for contacting the plant cell with the mutagenic chemical is at least about 5°C, 7°C, 10°C, 15°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 45°C, 47°C or 50°C. In addition or alternatively, a suitable temperature for contacting the plant cell with the mutagenic chemical is less than about 60°C, 57°C, 55°C, 50°C, 47°C, 45°C, 40°C, 38°C, 36°C, 34°C, 32°C, 30°C, 28°C, 26°C, 24°C, 22°C, 20°C, 17°C, 15°C or 10°C. In a further embodiment, the temperature for contacting the plant cell with the mutagenic chemical is about 5°C, 7°C, 10°C, 15°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 45°C, 47°C or 50°C.

In an embodiment, the cell is contacted with the mutagenic chemical at a temperature of about 5 - 50 °C, about 7 - 47 °C, about 10 - 45°C, about 15 - 40°C, about 16 - 35°C, about 17 - 30°C, about 18 - 28°C, about 19 - 26°C, about 18 - 25°C, preferably about 15 - 25°C or room temperature.

In a further embodiment, the cell is contacted with the mutagenic chemical at a temperature as detailed herein above, wherein this temperature is maintained during the period that the cell is contacted with the chemical. For example, if the cell is contacted with the mutagenic chemical at a temperature of about 20°C, this temperature of about 20°C is maintained during the full period that the cell is contacted with the mutagenic chemical. Preferably, the plant cell can be contacted with the mutagenic chemical at a temperature that is maintained at about 5 - 50 °C, preferably 15 - 25 °C, during the period that the cell is contacted with the chemical.

Alternatively, the temperature can increase and/or decrease during the period that the cell is contacted with the mutagenic chemical. For example, during the period that the plant cell is contacted with the mutagenic chemical, the temperature can increase about 1 °C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C. Alternatively, the temperature can decrease with about 1 °C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C. Hence as a non-limiting example, the starting temperature can be about 20°C and temperature can be about 22°C at the end of the period. Alternatively or in addition, the temperature can fluctuate during the time that the plant cell is contacted with the mutagenic chemical. For example, the temperature can fluctuate 1 °C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C during the period that plant cell is contacted with the mutagenic chemical.

After the plant cell is contacted with the mutagenic chemical under the conditions as detailed herein, the cell is preferably washed to remove the mutagenic solution. The cell can be washed at least 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. Preferably, the washing steps take in total about 10, 20, 50, 60, 80, 100 or 120 minutes.

In a further embodiment, the M. esculenta FEC cell is contacted for about 120 minutes with a mutagenic solution that comprises about 0.6% ENU. Preferably, the mutagenesis step takes place at room temperature, i.e. at about 20°C.

The cell can be washed with any suitable solution. Such suitable solutions are well-known to the person skilled in the art. The washing solution is for example, but not limited to, PBS or culture medium. A suitable medium for washing can e.g. be liquid GDS medium.

After the mutagenesis step and optional washing steps, the cell can be placed on an agar plate to recover. Such agar plates and culturing conditions are well-known for the person skilled in the ar. For example, but not limited to, the plant cell can be placed on GDc250 agar plates under suitable culturing conditions. Suitable culturing conditions are for example, but not limited to, about 28°C, about 16 hours light/about 8 hours dark and/or 3 Lux.

In a further optional step, the mutagenized cells are selected for a specific trait by culturing the mutagenized cells under specific, stringent conditions. As a non-limiting example, the culturing conditions of the mutagenized cell can include the presence of a herbicide, to select cells that have an increased herbicide resistance. A non-limiting example of a herbicide is an ALS (acetolactate synthase) inhibitor, such as imazapyr, flumetsulam or chlorsulfuron.

Other non-limiting examples of culturing conditions that result in the selection of mutagenized cells having a specific trait of interest include e.g. virus infection or stress conditions such as an increased drought or increased or decreased temperatures.

In a further embodiment, the method comprises a step of generating a cotyledonary embryo from the mutagenized cell. The terms“cotyledonary embryo” and“cotyledonary stage embryo” can be used interchangeably herein. The cotyledonary embryo in the method of the invention is preferably a cotyledonary stage somatic embryo. In a further embodiment, all cells of the cotyledonary embryo comprise the mutagenized DNA, preferably all cells of the cotyledonary embryo comprise the same mutagenized DNA.

The mutagenized cotyledonary embryo produced by the method of the invention can be further developed into a plantlet. The plantlet can further be developed into a plant. Hence in an embodiment of the invention, the method comprises a further step of generating a plantlet and/or a plant from the mutagenized M. esculenta cell. Preferably, the generated M. esculenta plant produces an edible starchy, tuberous root.

An embodiment of the invention further comprises detecting at least one mutation in the DNA of the mutagenized plant cell. Such mutation can be detected using any conventional means in the art. Detection of a mutation or mutations can be performed as e.g. described in W02005/021794, W02007/037678, W02006/137733, W02007/073165, and/or W02007/1 14693.

In one embodiment, the mutation is detected using DNA fingerprinting, an Oligonucleotide Ligation Amplification (OLA)-based assay and/or sequencing of the mutated DNA. A non-limiting example of an DNA finger-printing technique is AFLP. Alternatively or in addition, the DNA can be sequenced using conventional Sanger sequencing or using deep-sequencing technologies. Such deep-sequencing technologies are well-known in the art for the skilled person. Examples of deepsequencing technologies include, but are not limited to, Heliscope single molecule sequencing, Single molecule real time (SMRT) sequencing, Ion Torrent sequencing (ion semiconductor), 454 sequencing (pyrosequencing, Roche 454 Life SciencesTM, Branford, CT), Solexa (sequencing by synthesis, lllumina, Inc, San Diego, CA) and / or SOLD sequencing (sequencing by ligation, ABI, Applied Biosystems, Indianapolis, IN) and/or PACbio sequencing, e.g. the PACbio Sequel system.

The mutation can be detected by sequencing all mutated DNA molecules (e.g. the complete genome) or only part of the mutated DNA is sequenced (e.g. specific genomic fragments). In an embodiment, the mutation is detected as described in Rigola et al (2009), PLoS one, 4(3):e4761.

In a further embodiment, a phenotype can be assigned to the plant cell, embryo, plantlet or plant comprising the mutated DNA. Hence, the method can comprise a further step of detecting at least one mutation in the DNA and subsequently assigning a phenotype to the detected mutation. Alternatively, the method can comprise a step of detecting a phenotype and subsequently assigning at least one mutation in the DNA to the detected phenotype.

An assigned or detected phenotype is preferably a phenotype of interest, and the terms“a phenotype of interest” and“a trait of interest” can be used interchangeably herein. A plant cell, embryo, plantlet or plant having a phenotype of interest can be subsequently selected for e.g. propagation.

Exemplary, non-limiting traits of interest include yield, disease resistance, agronomic traits, abiotic traits, protein composition, oil composition, starch composition, insect resistance, fertility, silage, and morphological traits. In some embodiments, two or more traits of interest are screened for and/or against (either individually or collectively) in the plant cell, embryo, plantlet or plant comprising the mutated DNA.

Alternatively or in addition, the mutation causing the trait of interest can be determined and subsequently introduced in a further plant, such as a wild-type plant or a cultivar. The mutation can be introduced in a further M. esculenta plant, or in any other plant species. Non-limiting examples of other plant species include, for example, but not limited to, a member of Euphorbiaceae, which besides M. esculenta, can include Ricinus communis (castor oil plant), Jatropha curcas (Barbados nut), Hevea brasiliensis (Para rubber tree), Euphorbia pulcherrima (poinsettia), Euphorbia esula (leafy spurge) and Triadica sebifera (Chinese tallow). Similarly, the mutation discovered using the method of the invention as disclosed herein can be introduced e.g. in a member of the genus Manihot.

Alternatively or in addition, the mutation or mutations causing the trait of interest can be linked to a specific gene. Hence, using the method of the invention a specific gene can be assigned to a trait of interest in M. esculenta. In an optional further step of the invention, the gene that is linked to the trait of interest can be mutated in a further plant to increase or decrease its function. Similarly, a homolog of such gene can be mutated in other plant species to obtain the trait of interest.

In a second aspect, the invention pertains to a method for chemical mutagenesis of DNA in a Manihot esculenta cell, wherein the method comprises a step of contacting the cell with a mutagenic chemical. The mutagenic chemical preferably introduces a SNP in the DNA of the cell, wherein the mutation frequency is preferably as defined herein above. The mutagenic chemical is preferably a mutagenic chemical as described herein. Preferably, the mutagenic chemical is 1 -Ethyl-1 - nitrosourea (ENU). The Manihot esculenta cell is preferably a M. esculenta cell as defined herein. Preferably, the M. esculenta cell is a friable embryogenic callus (FEC) M. esculenta cell. Preferably, the DNA is chloroplast, mitochondrial or genomic DNA. Preferably, the DNA is genomic DNA.

Preferably, the cell is incubated in a mutagenic solution comprising the mutagenic chemical and wherein the concentration of the mutagenic chemical in the solution is preferably as defined herein above. Preferably, the mutagenic solution is a mutagenic solution as defined herein above. Preferably, the cell is contacted with the mutagenic chemical for a period as defined herein above. The cell is preferably contacted with the mutagenic chemical at a temperature as defined herein above. Preferably, the method further comprises a step of generating a cotyledonary embryo, plantlet and/or a plant from the mutagenized cell as defined herein above. The method further preferably comprises detecting at least one mutation in the DNA and optionally assigning a phenotype to the detected mutation as defined herein above.

In a third aspect, the invention pertains to a mutagenized cell, cotyledonary embryo, plantlet and/or plant that is obtainable by the method as defined herein. Preferably, the mutagenized cell, cotyledonary embryo, plantlet and/or plant is a mutagenized Manihot esculenta plant cell, cotyledonary embryo, plantlet and/or plant. Preferably, the plant cell, cotyledonary embryo, plantlet and/or plant is directly obtained from the method as defined herein. Hence, said mutagenized cell carries at least one or more SNPs in the DNA that was introduced by chemical mutagenesis of the parental plant.

Without wishing to be bound by theory, it was surprisingly observed that a single mutagenized FEC cell seems to ultimately develop into a plant. As a result, all cells of the cotyledonary embryo, plantlet and/or plant will thus comprise the same mutagenized genome. Therefore preferably, the cotyledonary embryo, plantlet and/or plant that is obtainable by the method as defined herein is not a chimeric cotyledonary embryo, plantlet and/or plant. Put differently, the mutagenized cell, cotyledonary embryo, plantlet and/or plant preferably comprises substantially, preferably only, one type of mutagenized genome.

In a fourth aspect, the invention concerns a composition comprising i) a mutagenic solution comprising the mutagenic chemical 1 -Ethyl-1 -nitrosoure and ii) a Manihot esculenta cell. Preferably the Manihot esculenta cell is a M. esculenta plant cell as defined herein above. The mutagenic solution is preferably a mutagenic solution as defined herein.

In a fifth aspect, the invention pertains to the use of the mutagenic chemical 1 -Ethyl-1 -nitrosourea for the chemical mutagenesis of the DNA in a plant cell, preferably a Manihot esculenta cell. The mutagenic chemical is preferably used in a method as defined herein above.

In a sixth aspect, the invention pertains to the progeny of the mutagenized cell, cotyledonary embryo, plantlet and/or plant that is obtainable by the method of the invention. Hence, the progeny carries at least one or more SNPs in the DNA that was introduced by chemical mutagenesis of the parental plant.

In a further aspect, the invention concerns plant parts that are derived from the mutagenized plant of the invention, or are derived from the progeny of the mutagenized plant. Preferably, the plant parts do not constitute propagation material. Preferred plant parts are the cassava roots, or the products derived therefrom. Derived products include, but are not limited to, nutritional products (e.g. for human or animal consumption), alcoholic beverages, biofuel, laundry starch and/or medicinal use. Preferably, said plant parts, non-propagating material and/or products derived therefrom comprise at least one or more SNPs in the DNA that was introduced by chemical mutagenesis of the parental plant.

Description of the figures

Figure 1. The effect of increasing mutagen concentration and toxicity on cotyledonary embryo regeneration from FECs. The total number of cotyledons recovered from mutagen-treated 60444 FECs is shown. A) EMS (ethyl methanesulfonate), B) MMS (methyl methanesulfonate), C) NaN3 (sodium azide) D) ENU (N-ethyl-N-nitrosourea), E) AzaC (azacytidine) and F) NQO (4- nitroquinoline 1 -oxide). # indicates mutagen concentrations that resulted in visibly reduced FEC growth which can increase (e.g. EMS), reduce (e.g. NaN3) or prevent (e.g. NQO) regeneration of cotyledonary embryos from treated FECs. All results shown were obtained from cassava variety 60444. Figure 2. Mutation Breeding sequencing results. Percentage of cassava plantlets containing mutations (A) and mutation frequency (B). Mutation spectrum obtained for EMS (C) and ENU (D). EMS - ethyl methanesulfonate; MMS - methyl methanesulfonate; NaN3 - sodium azide; ENU - N- ethyl-N-nitrosourea; AzaC - azacytidine; NQO - 4-nitroquinoline 1-oxide. All results shown were obtained from cassava variety 60444.

Figure 3. ENU treatment of TME7 FECs. Base changes were detected by PACbio sequencing. 3A) number and type of base changes in 1 13 cassava TME7 plants regenerated from FECs treated with 0.1 % ENU for 2 hours, 3B) number and type of base changes in 17 cassava TME7 plants regenerated from FECs treated with 0.3% ENU for 2 hours, 3C) number and type of base changes in 332 cassava TME7 plants regenerated from FECs treated with 0.6% ENU for 0.5 hours, 3D) number and type of base changes in 1 1 1 cassava TME7 plants regenerated from FECs treated with 0.6% ENU for 1 hour, 3E) number and type of base changes in 17 cassava TME7 plants regenerated from FECs treated with 0.6% ENU for 2 hours, 3F) number and type of base changes in 146 cassava TME7 plants regenerated from FECs treated with 1 % ENU for 0.5 hours, and 3G) number and type of base changes in 40 cassava TME7 plants regenerated from FECs treated with 1 % ENU for 1 hour.

Figure 4. The mutation rate and number of mutations in ENU-treated TME7 FECs. Base changes were detected by PACbio sequencing 4A) the mutation rate (per kb) for all types of base changes in 776 cassava TME7 plants regenerated from FECs treated with ENU, per treatment, 4B) number of mutations for all types of base changes in 776 cassava TME7 plants regenerated from FECs treated with ENU, per treatment.

Figure 5. ENU treatment of 60444 FECs. Base changes were detected by PACbio sequencing. 5A) number and type of base changes in 79 cassava 60444 plants regenerated from FECs treated with 0.1 % ENU for 2 hours, 5B) number and type of base changes in 41 cassava 60444 plants regenerated from FECs treated with 0.6% ENU for 0.5 hours, 5C) number and type of base changes in 26 cassava 60444 plants regenerated from FECs treated with 0.6% ENU for 1 hour.

Figure 6. The mutation rate and number of mutations in ENU-treated 60444 FECs. Base changes were detected by PACbio sequencing. 6A) the mutation rate (per kb) for all types of base changes in 146 cassava 60444 plants regenerated from FECs treated with ENU, per treatment and 6B) the number of mutations for all types of base changes in 146 cassava 60444 plants regenerated from FECs treated with ENU, per treatment. Examples

Example 1

Method

Friable embryogenic calli (FECs) of cassava variety TMS60444, also named herein 60444 or “60444” are generated and multiplied according to the protocols published by Bull et al. (2009) or Chauhan et al. (2015). Up to 40 FEC clusters (approximately 3 weeks after multiplication are transferred to a single 50 ml_ Falcon tube and GDS liquid (appropriate for the final desired ENU concentration) is added. FECs in liquid are vortexed to ensure FECs are broken apart from the clusters. N-nitroso-N-ethylurea (ENU, Sigma Aldrich, N8509-5G) is dissolved by resuspending, then vigorous hand-shaking, in GDS liquid for a maximum of 20 minutes to create a 1 % stock solution. 1 % ENU stock solution is added to the FECs in liquid to generate the desired ENU concentration, up to a final volume of 25 ml_. FECs are incubated with ENU on a rotating shaker (20 rpm) for up to 2 hours.

Tubes are removed from the shaker, and FECs are allowed to settle to the bottom of each tube before as much supernatant as possible (without disturbing the FECs) is removed and discarded. After removal of the supernatant, the final volume of FECs and liquid combined should preferably be no more than 5 ml_. 30 ml_ of GDS liquid (liquid GDS medium) is added to each tube and hand-shaken to mix well. An additional seven wash steps are performed as described above (supernatant discarded, GDS liquid added and shaken well) over a minimum of 2 hours. If necessary, FECs are left to soak in the sixth wash solution to extend the washing time to at least 2 hours. FECs are then evenly and sequentially split between tubes to allow the equivalent of five FEC clusters to be evenly spread at once across five mesh circles (as described by Bull et al. (2009)). FECs (on mesh) are placed on GDc250 agar plates, sealed with parafilm and allowed to recover from mutagenesis for 1 week (28°C, 16 hours light/8 hours dark, 3 Lux). FECs (on mesh) are then transferred to MSNc250 agar plates (with or without herbicide selection) to allow regeneration of cotyledonary embryos (28°C, 16 hours light/8 hours dark, 3 Lux). Transfer to MSNc250 agar plates (with or without herbicide selection) is repeated weekly for up to 11 weeks. Regenerated cotyledonary embryos are transferred on a weekly basis to fresh CEMd OO agar plates (for up to 8 weeks) to promote establishment and elongation of shoot apical meristems. Shoots are then transferred to CBMc50 jars to allow development of plantlets which can be sequenced, further multiplied in tissue culture and/or transferred to the greenhouse.

Materials

All media types are described in Bull et al. (2009); specifically GDS liquid (without carbenicillin), GD + C250 solid medium, MSN + C250 medium, CEM + C100 solid medium, CBM + C50 medium.

Results

Six mutagens were tested for their suitability for mutagenesis of cassava FECs, specifically ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), sodium azide (NaN3), N-ethyl-N- nitrosourea (ENU), azacytidine (AzaC) and 4-nitroquinoline 1-oxide (NQO). Mutagen toxicity was identified as a key factor for successful regeneration of mutated cassava plantlets. Choice of mutagen is crucial for mutation density and spectrum. Length of exposure to the mutagenic chemical will be assessed to determine if the balance between low toxicity and sufficiently high mutation density can be further optimised to ensure high rates of mutant production.

Figure 1 illustrates the effect of increasing mutagen concentration and toxicity on cotyledonary embryo regeneration from FECs. FECs were sensitive to mutagen toxicity caused by four out of six mutagens at the concentrations tested. All tested concentrations of NQO (0.01 % and above), as well as MMS concentrations of 0.3% and above, were entirely toxic to FECs, preventing any FEC growth or cotyledonary embryo regeneration. Higher concentrations of NaN3 (2.5% and 5%) had a visibly negative effect on FEC growth and also resulted in reduced embryo regeneration. Results obtained for the highest concentrations of ENU (1 % and 1.3%) and AzaC (1 %) suggest that high concentrations of these mutagens can also promote reduced embryo regeneration. Nevertheless, The effect of ENU toxicity on 60444 FECs seems to be minimal and significantly less than EMS, MMS, NQO and NaH3. The highest tested concentration of EMS (2%) negatively impacted FEC growth, yet conversely promoted significantly higher levels of embryo regeneration than all other EMS concentrations and the untreated control. Moderately high concentrations of MMS (0.1 %) and ENU (0.1 % - 0.6%) also appeared to have a positive effect on embryo regeneration. The positive effect of EMS 2% on cotyledonary embryo regeneration, despite reduced FEC growth, indicated that FEC plating density may be correlated with embryo regeneration rate. This effect has been observed in subsequent experiments where various FEC plating densities were tested (without chemical mutagenesis). In particular, these subsequent experiments showed that reducing FEC density of non-mutagenised FECs resulted in increased cotyledon regeneration. Hence, without wishing to be bound by theory, a reduced FEC density due to toxicity could subsequently result in the increased cotyledon regeneration that was observed for the 2% EMS treatment (Figure 1 A).

Cassava plantlets regenerated from FECs subjected to chemical mutagenesis were sequenced using KeyPoint® Mutation Breeding (as taught in Rigola et al, supra), allowing determination of the identity and location of SNPs across a total of 60,585 bp sequenced in each plant (Figure 2). Very few SNPs were identified in NaN3- and AzaC-treated plantlets, whereas higher number of plantlets treated with ENU, EMS and MMS contained SNPs. The mutation spectrum created by ENU was broader than the mutations produced by EMS treatment.

Example 2

Method

Friable embryogenic calli (FECs) of variety TME7 and 60444 are generated and multiplied according to the protocols published by Bull et al. (2009) or Chauhan et al. (2015). Up to 40 FEC clusters (approximately 3 weeks after multiplication) are transferred to a single 50 mL Falcon tube and GDS liquid (appropriate for the final desired ENU concentration) is added. FECs in liquid are vortexed to ensure FECs are broken apart from the clusters. N-nitroso-N-ethylurea (ENU, Sigma Aldrich, N8509-5G) is dissolved by resuspending, then vigorous hand-shaking, in GDS liquid for a maximum of 20 minutes, preferably 1 to 20 minutes, to create a 1 % stock solution. 1 % ENU stock solution is added to the FECs in liquid to generate the desired ENU concentration, up to a final volume of 25 ml_. These concentrations are 0.1 %, 0.3%, 0.6% and 1 % (weight of ENU per volume of final reaction mixture). FECs are incubated with ENU on a rotating shaker (20 rpm) for either 0.5 hours, 1 hour or 2 hours. In all treatments on TME7 FECs, the number of clumps of FECs used is the same, thus the number of FECs exposed to the mutagenic chemical is approximately the same for each treatment. In all treatments on“60444” FECs, the number of clumps of FECs used is the same, thus the number of FECs exposed to the mutagenic chemical is approximately the same for each treatment.

Washing steps along with subsequent cultivation and regeneration are performed as in Example 1.

Induced mutations are detected by sequencing. This is achieved by isolation of genomic DNA from individual regenerant plants using a CTAB isolation protocol familiar to those skilled in the art, followed by PCR amplification of amplicons from each DNA sample individually. From each plant more than 40 amplicons are produced, covering almost 130kb of sequence in total. These amplicons are pooled according to their length, keeping similar length amplicons in the same pool. Seven pools are produced from the approximately 800 TME7 samples and two pools from the approximately 160 “60444” samples. Pooled amplicons derived from each individual plant are tagged by performing a PCR amplification using primers with an extension tail having a sequence unique within the pool. All amplicons in a pool are then combined and purified to remove short DNA fragments such as unused primers and primer dimers. The amplicons in each pool are sequenced using the PACbio Sequel platform. Each pool is sequenced on a separate SMRTCell.

Materials

All media types are described in Bull et al. (2009); specifically GDS liquid (without carbenicillin), GD + C250 solid medium, MSN + C250 medium, CEM + C100 solid medium, CBM + C50 medium.

Results

N-ethyl-N-nitrosourea (ENU) was used to mutagenize FECs of cassava varieties TME7 and 60444. The concentration of ENU and length of exposure to the ENU solution has been assessed to determine the optimal conditions to allow for high plantlet regeneration rates and sufficiently high mutation density.

Figure 3 shows the number and type of mutations detected in the TME7 plantlets regenerated from the specific treatment indicated in the figure legend. Figure 4A shows the total mutation rate in mutations per kilobase of sequence per treatment in regenerated TME7 plants. Figure 4B shows the total number of mutations detected in the plants regenerated from each treatment.

Figure 5 shows the number and type of mutations detected in the“60444“ plantlets regenerated from the specific treatment indicated in the figure legend. Figure 6A shows the total mutation rate in mutations per kilobase of sequence per treatment in regenerated “60444” plants. Figure 6B shows the total number of mutations detected in the plants regenerated from each treatment.

The distribution of mutation types (base changes) caused by ENU appears to be largely independent of the treatment conditions within the TME7 plants regenerated. Changes from the purine nucleotides adenine and thymine are predominant, however all base changes are detected including the pyrimidine changes cytosine to guanine and guanine to cytosine.

In the TME7 plants the average mutation rate for all base changes varies per treatment between about 0.004 per kb and about 0.007 per kb. The largest number of mutations detected is from the 0.6% ENU treatment for 0.5 hours. This treatment allows the largest number of plants to regenerate whilst causing a useful mutation rate. The toxicity of ENU and its breakdown products becomes more acute with treatments above 1 % ENU and or with treatment durations longer than 0.5 hours. The optimal treatment of ENU on TME7 FECs is about 0.6% for about 0.5 hours. The distribution of mutation types (base changes) detected within the“60444” plants regenerated from different treatment conditions is not as uniform as with TME7 plants. However, since the number of“60444” plants regenerated and thus mutations detected is considerably less than with TME7 plants, the distribution pattern may be more variable. All base changes are detected in regenerated “60444” plants, including pyrimidine changes cytosine to guanine and guanine to cytosine, as for TME7.

In the regenerated“60444” plants the average mutation rate for all base changes varies per treatment between about 0.003 per kb and about 0.006 per kb. The largest number of mutations detected is from the 0.1 % ENU treatment for 2 hours. This treatment allows the largest number of plants to regenerate. The optimal treatment of ENU on“60444” FECs is about 0.1 % for about 2 hours. A treatment of 0.3% ENU for 2 hours on“60444” FECs resulted in the regeneration of only two plants, suggesting that cassava variety 60444 is more sensitive to the toxicity effect than variety TME7.

The results are summarized in the Table below:

Table 1A. ENU treatment of TME7 FECs

Table 1B. ENU treatment of 60444 FECs

Conclusion

ENU causes all possible base changes in cassava DNA, with a preference for changing the purine nucleotides adenine and thymine. Although ENU appears to be less toxic than several other mutagens, the toxicity effect can be reduced further by careful moderation of the duration of exposure and the concentration of ENU used in the treatment solution. For cassava variety TME7, the optimal treatment is about 0.6% for about 0.5 hours. This treatment gives a mutation rate of about 0.006 mutations per kilobase, which allows the generation of a useful number of mutations in target genes without causing a background mutation rate which might be detrimental. Further, this treatment is mild enough to allow the regeneration of a large number of plants and thus results in an optimal yield of mutations in any specific gene.

For cassava variety 60444, the optimal treatment is about 0.1 % for about 2 hours; the concentration of ENU may be increased if the treatment duration is decreased. This treatment gives a mutation rate of about 0.006 mutations per kilobase, which allows the generation of a useful number of mutations in target genes without causing a background mutation rate which might be detrimental. Further, this treatment is mild enough to allow the regeneration of a large number of plants and thus results in an optimal yield of mutations in any specific gene.